Multiple Wnt family members are expressed in overlapping patterns in development and in adult tissues. For example in human mammary tissue Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b, and Wnt10b are expressed during breast development and in some breast tumors Wnt2, Wnt4, Wnt5A, Wnt7B, Wnt10B, and Wnt13 have been shown to be overexpressed. Although Wnt signaling is critical to embryonic development, stem cell maintenance, differentiation and metastasis, no studies have considered the possibility of the interaction of family members. Recently, we showed a novel aspect of Wnt biology, that Wnt11 and 5a proteins can functionally and physically interact with each other. However, little is known about the mechanism or biological significance of Wnt/Wnt protein interaction. The aims of this project are to answer the questions: Specific Aim 1: What is the mechanism of Wnt11/5a interaction? Based on preliminary evidence that depleting the tyrosyl sulfur transferase enzyme TPST1 inhibits the interaction of Wnt5a and 11 in co- immunoprecipitation experiments, we hypothesize that they interact non-covalently via tyrosine sulfated amino acids. Specific Aim 2: What is the biological significance of Wnts interacting with Wnts? We have shown in published work that both Wnt11 and 5a are required for the establishment of the dorsal axis in Xenopus, and in preliminary data that secreted Wnt11/5a complexes are more active in canonical Wnt pathway signaling than each Wnt secreted alone. Here we will test the hypothesis that Wnt11/5a complexes have different signaling effects on mouse L cells and on human ES cells than each Wnt acting alone. We will also test the hypothesis that Wnt/Wnt complexes are important in ventral mesoderm formation (zygotic Wnt8/ maternal 5a), in establishing the heart field (Wnt 6/ maternal Wnt5a), and in posterior endoderm formation (zygotic Wnt11/5a). PUBLIC HEALTH RELEVANCE: The Wnt family of signaling molecules are known to be essential for patterning the embryo, for cell differentiation and are known to be misregulated in human malignancies including colon and breast cancer. We recently discovered a novel aspect of Wnt biology, that subfamily members interact with each other both physically and functionally. In this study we aim to determine the mechanism of interaction and the broader significance of Wnt/Wnt complex formation in Xenopus, mouse and human cells.